Light-microscopic imaging methods have been recently developed that have made it possible to image sample structures smaller than the diffraction-induced resolution limit of conventional light microscopes by employing a sequential, stochastic localization of individual markers, in particular fluorescence molecules. Such methods are described, for example, in WO 2006/127692 A2, DE 10 2006 021 317 B3, WO 2007/128434 A1, U.S. 2009/0134342 A1, DE 10 2008 024 568 A1, WO 2008/091296 A2, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nature Methods 3, 793-796 (2006), M. J. Rust, M. Bates, X. Zhuang; “Resolution of Lambda/10 in fluorescence microscopy using fast single molecule photo-switching,” Geisler C. et al, Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also referred to as localization microscopy. The applied methods are known in the literature, for example, by the designations (F)PALM ((fluorescence) photoactivation localization microscopy), PALMIRA (PALM with independently running acquisition), GSD(IM) (ground state depletion individual molecule return) microscopy) or (F)STORM ((fluorescence) stochastic optical reconstruction microscopy).
The new methods have in common that the preparation of sample structures to be imaged involves using point-like objects, generally referred to as markers, that have two distinguishable states, namely a “bright” state and a “dark” state. For example, if fluorescent dyes are used as markers, then the bright state is a state capable of fluorescence, and the dark state is a state that is incapable of fluorescence.
In specific embodiments, for example, in WO 2008/091296 A2 and WO 2006/127692 A2, photoswitchable or photoactivatable fluorescence molecules are used. Alternatively, as, for example, in the German Examined Accepted Specification DE 10 2006 021 317 B3, inherent dark states of standard fluorescence molecules are utilized.
To image sample structures at a resolution higher than the conventional resolution limit of the imaging optics, a small subset of the markers is repeatedly transferred to the bright state. This requires selecting a density of the markers forming this active subset that will ensure that the average distance between adjacent markers in the bright state and, thus, in the state that can be imaged by light microscopy, is greater than the resolution limit of the imaging optics. The markers forming the active subset are imaged onto a spatially resolving light detector, for example, a CCD camera, so that a light distribution in the form of a light spot, whose size is determined by the resolution limit of the optics, is recorded from each point-like marker.
A multitude of individual raw data images are thereby captured in each of which another active subset is imaged. In each individual raw data image, the centroid positions of the light distributions, that represent the point-like markers in the bright state, are then determined in an image analysis process. The centroid positions of the light distributions ascertained from the individual raw data images are then brought together in an overall representation in the form of an assembled image data record. The highly resolved assembled image resulting from this overall representation reflects the distribution of the markers.
Representatively reproducing the sample structure to be imaged requires that a sufficient number of marker signals be detected. However, since the number of markers in the particular active subset is limited by the required minimal average mutual distance between two markers in the bright state, a very large number of individual raw data images must be recorded to be able to completely image the sample structure. The number of individual raw data images typically ranges from 10,000 to 100,000.
In the above described localization microscopy methods, the centroid positions of the individual light spots produced on the light detector are typically only determined in two dimensions, so that the highly resolved image of the sample structure reconstructed from all of the centroid positions is likewise merely a two-dimensional image. It would be preferable, however, to reconstruct the sample structure in three dimensions.
Some related art methods make possible a three-dimensional positional determination of individual point-like objects in a sample. In this regard, reference is made exemplarily to WO 2009/085218 A1 and U.S. Pat. No. 7,772,569 B2, as well as to “Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging,” Optics Letters, 2003, vol. 28 (no. 2), Michael Speidel et al; and “Three-dimensional Particle Tracking via Bifocal Imaging,” Nano Letters, 2007, vol. 7 (no. 7), pages 2043-2045, Erdal Toprak, et al. The method described in U.S. Pat. No. 7,772,569 B2 is based on the analysis of defocused image patterns on at least two image sensors in different image planes. In the method according to WO 2009/085218 A1, ellipticities of light patterns are analyzed using special optics.
The related art methods require a complicated'image analysis for centroid determination and pattern recognition, or special optics that limit the general usefulness of microscopic devices functioning in accordance with these methods.